Hi Everyone!
I'm trying to assay the binding activity of my protein against a cytotoxic protein. . I can do it in two ways, first by rescue assay of mammalian cell line and second by ELISA. However, I have polyclonal antibodies against my protein. therefore, will ELISA be a reliable method for such study?
Dear Ravinder,
ELISA is reliable in most cases.
But since you are looking for the binding activity of your protein of interest against a cytotoxic protein.
Option 1.
It is better to use a commercial cytotoxic protein specific monoclonal antibody as a capture antibody.
All you need is a standard cytotoxic protein and a monoclonal antibody to capture it.
then you can probe your protein to have a binding activity.
Finally you can measure the binding activity by probing your primary polyclonal antibody conjugated to HRP or FITC specific to your protein of interest.
This quite lengthy protocol.
Option 2.
Immobilize your histagged commercial cytotoxic receptor protein on a Ni-NTA biosensor chip. (BIACore assay)
Then probe your cytotoxic protein followed by your purified protein of interest to study interaction between your cytotoxic protein and your protein of interest.
you can determine the affinity value.
If you do not have much experience with ELISA and want to set up an assay based on your antibodies, it's a risky business. There are several options:
1. Buy a commercial kit (already mentioned above) and strictly adhere to the instructions! Look on the data sheet before you purchase it. If it is carefully validated, it should work in most cases.
2. Try to find an immunoassay expert in your university and establish a collaboration. This is the best option, if you want to use your own antibodies.
3. Try to set up your own assay based on text books and papers. This might need some time and perhaps you need to make an new, improved antibody.
Dear Ravindra Singh Rawat
ELISA is reliable. You better use monoclonal ABs. good luck
Regards
Prof. Houda Kawas
Dear ELISA is reliable if you follow the manufacturer's instructions carefully and correctly.
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